This research plan is to develop a Microcarrier-Bioreactor Cell Culture System for the growth and efficient recovery of infected, whole cells from microcarriers in controlled, bioreactor cultures. Primary chick embryo fibroblast (CEF) cells are widely used in viral vaccine production (for both avian and human vaccines), but there are no known licensed-manufacturing protocols which utilize a Microcarrier-Bioreactor System for this purpose. Focus will be on the production of a whole cell Marek's Disease vaccine for avian applications. The System technology developed in the course of these studies could replace large-scale industrial processes in which (1) primary CEF cells are utilized in roller bottles, (2) embryonated, fertile eggs function as tiny bioreactors, such as in the manufacturing of human influenza vaccine or (3) whole cell vaccines and/or immunotherapies. Specific Aims I, II and VI. To optimize cell culture conditions for a one to two day HVT-production run using primary CEF cells, secondary CEF cells and immortalized CEF-like cells at high density, in a one-liter bioreactor culture. Specific Aim III. To optimize culture conditions for a three-day HVT-production run using secondary CEF cells at the one-liter scale in bioreactor culture. Specific Aim IV & V. To develop protocols for rapid harvesting of HVT-infected cells, and separation of those cells from microcarriers, and to achieve high-titer Marek's vaccine production. PROPOSED COMMERCIAL APPLICATION: Many animal vital vaccines (e.g. Marek's) and human viral vaccines are produced using chick embryo cells, either in roller bottle systems or in embryonated chicken eggs. Neither process uses microcarrier - bioreactor systems. Large scale production of Marek's vaccine in a Miaocarrier-Bioreactor System, will lead to substantial economic advantages when bioreactors are compared to old technology. Product quality and consistency should also be improved. and manufacturing costs reduced. The System herein proposed will utilize (1) primary or secondary CEF cells, or immortalized cell lines. (2) special media formulations, (3) bovine sera and will evolve the (4) protocols to produce vaccines in this System. The technology should extrapolate to other whole-cell human vaccines. PROPOSED COMMERCIAL APPLICATION: The current short supply of human influenza vaccine serves to underline the importance for developing technology that is an alternative to embryonated egg production methods. The lead time from (1) a CDC estimate of the specific influenza virus variant likely to strike the USA, to (2) the availability of the vaccine on a large scale is exceedingly long, many months. In the event of an influenza pandemic this many-month lead-time could result in a high fatality rate reminiscent of the 1917-18 pandemic. If influenza virus vaccine can be produced in very large-scale bioreactors, with a fast turn around time (a couple months), the high fatality rate of a pandemic might be averted. The research proposed has the potential to address this issue.